Abstract
RNase Y is a key endoribonuclease affecting global mRNA stability in Bacillus subtilis. Its characterization provided the first evidence that endonucleolytic cleavage plays a major role in the mRNA metabolism of this organism. RNase Y shares important functional features with the RNA decay initiating RNase E from Escherichia coli, notably a similar cleavage specificity and a preference for 5′ monophosphorylated substrates. We used high-resolution tiling arrays to analyze the effect of RNase Y depletion on RNA abundance covering the entire genome. The data confirm that this endoribonuclease plays a key role in initiating the decay of a large number of mRNAs as well as non coding RNAs. The downstream cleavage products are likely to be degraded by the 5′ exonucleolytic activity of RNases J1/J2 as we show for a specific case. Comparison of the data with that of two other recent studies revealed very significant differences. About two thirds of the mRNAs upregulated following RNase Y depletion were different when compared to either one of these studies and only about 10% were in common in all three studies. This highlights that experimental conditions and data analysis play an important role in identifying RNase Y substrates by global transcriptional profiling. Our data confirmed already known RNase Y substrates and due to the precision and reproducibility of the profiles allow an exceptionally detailed view of the turnover of hundreds of new RNA substrates.
Highlights
In prokaryotic cells, mRNA half-lives range from seconds to over an hour
In E. coli, mRNA decay is primarily initiated through endonucleolytic cleavage by the single-strand specific RNase E at one or more internal sites, followed by rapid 39 exonucleolytic degradation of the decay intermediates [1]
Evidence that endonucleolytic cleavage plays a major role in mRNA metabolism in B. subtilis came from the initial characterization of RNase Y which showed a strong effect on global mRNA stability in strains depleted for this enzyme [12]
Summary
MRNA half-lives range from seconds to over an hour. Controlling the life span of a messenger can be an efficient means to control gene expression. Despite its central role in mRNA decay in E. coli, RNase E is absent from many eubacteria including the Gram positive model bacterium B. subtilis This organism instead possesses a pair of paralogous RNases, J1 and J2, which combine an RNase E-like endonucleolytic [5] and a 59 exonucleolytic activity that requires a 59 monophosphate group [6,7]. In a double mutant (expressing no RNase J2 and strongly reduced levels of RNase J1) global mRNA stability is only slightly increased [10], a finding not compatible with a key role for RNase J in initiating mRNA decay on a global scale that would be comparable with the role of RNase E in E. coli In extrapolation, this suggests that the endonucleolytic activity of RNases J1/J2 as well as the 59 P conversion of native transcripts by B. subtilis RppH occurs on a more limited scale
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